Method for preparing protease beverage by fermenting cyperus esculentus powder and product thereof

Abstract

The invention provides a method for preparing a protease beverage by fermenting cyperus esculentus powder and a product thereof. Comprising the steps of directional pretreatment, fermentation medium preparation, segmented fermentation and optional blending, specifically, directional pretreatment adopts low-temperature extrusion at the temperature of 60-70 DEG C and ultrasonic treatment at the power of 200-300 W for cooperative treatment, and the fiber structure and antinutritional factors of soybean meal are effectively broken; in the fermentation stage, lactobacillus plantarum, saccharomyces cerevisiae and bacillus coagulans composite flora are inoculated, anaerobic fermentation is performed at the temperature of 35-37 DEG C, the pH value of the system is reduced to 4.0-4.5, and then L-cysteine hydrochloride-vitamin B synergist is added for secondary fermentation in a micro-aerobic environment at the temperature of 38-40 DEG C. The fermentation process provided by the invention is simple, the treatment is convenient, the used equipment is conventional equipment in the food industry, and the method is suitable for large-scale industrial production. Meanwhile, in the prepared fermentation liquor, the protease activity is larger than or equal to 320 U / mL, the total number of viable bacteria is larger than or equal to 1.2 * 10 CFU / mL, the optimum pH of the protease is 3.8-4.8, the optimum temperature is 37-39 DEG C, the protease is highly matched with the human gastrointestinal tract environment, and the method has wide industrialization prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of resource utilization of chufa, and particularly relates to a method for preparing a protease beverage from chufa powder and a product thereof. BACKGROUND

[0002] Cyperus esculentus L. is a plant of Cyperus L. in Cyperaceae, which is a positive plant and likes light. It can adapt to various harsh growth environments, such as drought, waterlogging, barrenness, and salinity. It can be planted in Inner Mongolia, Liaoning, Guangdong, Guangxi, Fujian, Xinjiang, Gansu and other regions in China. Cyperus can be cultivated as an annual crop in production, and mainly uses the underground tubers of chufa (also known as underground almond, iron water chestnut, underground almond, underground chestnut, underground walnut, ginseng fruit and ginseng bean).

[0003] Chufa is an economic crop with high quality, high yield and broad prospects for comprehensive utilization, which integrates grain, oil, pasture and feed. It is not only a good oil crop, but also a good feed for livestock. It can be developed and planted as a good forage. In addition, it can also be used as lubricating oil and soap. Chufa is rich in nutrients such as oil, sugar, protein, vitamins and minerals. Chufa protein contains 18 kinds of amino acids, and the total amino acid content is 29.88%, of which essential amino acids account for 46.03%. The comprehensive nutrition of chufa is higher than that of corn and wheat. Chufa has high nutritional value and health care function, and is especially suitable for patients with diabetes and digestive system diseases. At the same time, chufa also has certain effect on preventing heart disease. In addition, chufa industry development has obvious advantages in ensuring national grain and oil safety, realizing oil, grain and feed self-sufficiency, agricultural supply side reform and other aspects, and has extremely important economic value and social value. It also has important ecological value in wind prevention and sand fixation. In recent years, a large number of documents have been issued by relevant departments to promote chufa planting and industry development. Therefore, chufa industry has great development space.

[0004] At present, the processing method of chufa mainly focuses on cold pressing oil process. Although this process can maximize the retention of nutritional ingredients of oil, it will produce a large amount of by-product, chufa meal. Chufa meal contains a certain amount of crude fiber and anti-nutritional factors (such as phytic acid, etc.), and if it is directly used as food raw material, the taste is rough and the nutritional absorption efficiency is low. Although the existing pretreatment methods (such as high temperature cooking and chemical soaking) can improve some problems, they are easy to cause loss of nutritional ingredients and introduction of chemical residues, etc. Therefore, in the prior art, chufa meal is mostly directly used as feed or fertilizer, and there is a lack of utilization method for food processing.

[0005] Meanwhile, the current consumer demand for functional drinks is growing, especially for drinks rich in active enzymes (such as protease), because they can assist protein digestion and absorption and improve intestinal function, and have broad market prospects. The preparation of existing protease drinks mostly uses soybean, milk and the like as raw materials, and has problems such as high raw material cost and complex process; moreover, single-strain fermentation is mostly used for fermented drinks, resulting in low enzyme activity, single nutritional components and insufficient utilization rate of raw materials. For example, when the prior art uses soybean meal as a raw material to prepare a fermented drink, the protease activity is often less than 100 U / mL due to improper strain selection or poor fermentation condition control, and the anti-nutritional factors in the product are not completely removed, affecting the safety and palatability of the drink.

[0006] Therefore, how to convert cyperus esculentus meal into a high-activity protease drink through a green and efficient process to achieve the dual goals of high-value utilization of by-products and development of functional drinks has become a technical problem to be solved by those skilled in the art. SUMMARY

[0007] The purpose of the present application is to provide a method for preparing a protease drink from cyperus esculentus powder and a product thereof. Through low-temperature extrusion and ultrasonic synergistic pretreatment, the structure is first broken at 60-70℃, and then 200-300W ultrasonic is used for solubilization, which effectively dissociates the crude fiber, destroys the phytic acid molecular structure and improves the xylan solubility, and does not need to add exogenous enzymes, avoiding the problem of enzyme residues. By using a specific complex bacterial population of food grade, a corresponding two-stage temperature control fermentation process and an enzyme activity enhancer, the protease activity and total number of viable bacteria at the end of fermentation are effectively improved, and the protease has an optimal pH (3.8-4.8), an optimal temperature (37-39℃), a gastric activity retention rate of 65% and a small intestinal activity retention rate of 88%, which is highly adapted to the human gastrointestinal environment.

[0008] To achieve the above-mentioned purpose, the present application provides a method for preparing a protease drink from cyperus esculentus powder, which specifically comprises the following steps:

[0009] (1) Directional pretreatment:

[0010] Select the meal of cyperus esculentus within 48h after cold pressing to extract oil, and after ultrafine grinding, add deionized water to perform low-temperature extrusion and ultrasonic synergistic treatment to obtain a meal pretreatment liquid;

[0011] (2) Preparation of fermentation medium:

[0012] In each 100mL of the meal pretreatment liquid obtained in step (1), add 2-3g of skimmed milk powder, 1-1.2g of glucose and 0.1-0.2g of KH2PO4, adjust the pH to 6.0-6.5, sterilize and then cool to 35-38℃;

[0013] (3) Subsection fermentation:

[0014] Inoculate the compound bacterial population into the fermentation medium prepared in step (2) at 5-8%, pass nitrogen gas in the first stage, and carry out anaerobic fermentation at 35-37°C for 18-24h to reduce the pH of the system to 4.0-4.5; in the second stage, raise the temperature to 38-40°C, pass sterile air containing 5-8% oxygen, and add enzyme activity enhancer at 0.1-0.2 g / 100 mL, and continue to ferment for 12-16h. After fermentation is completed, collect the fermentation liquor to obtain the product.

[0015] In a preferred embodiment, step (3) is further followed by step (4) of blending:

[0016] Centrifuge the fermentation liquor obtained in step (3), filter the supernatant through a 0.22μm organic filter membrane; add 0.5-1.0 g / 100 mL fructooligosaccharide and 1-1.5 g / 100 mL erythritol, homogenize at 20-25MPa and 40-45°C for 2-3 times, instantaneously sterilize at 135°C for 3-5s, and complete aseptic filling within 10s, and store at 2-8°C to obtain the protease beverage.

[0017] In a preferred embodiment, in step (1), the process parameters for cold pressing of the oil of Eleocharis dulcis are: pressing temperature ≤60°C and pressing pressure 30-40Mpa; the obtained soybean meal has water content ≤12%, protein content 5-15%, and purity ≥99.0%. The above parameters can avoid denaturation of the protein in the soybean meal and ensure the efficiency of subsequent physical treatment and fermentation.

[0018] In a preferred embodiment, in step (1), the particle size after ultramicro-pulverization is 80-120 mesh.

[0019] In a preferred embodiment, in step (1), the solid-liquid ratio of the soybean meal to deionized water is 1g:(1-12)mL; preferably, the solid-liquid ratio of the soybean meal to deionized water is 1g:(8-10)mL.

[0020] In a preferred embodiment, in step (1), the low-temperature extrusion and ultrasonic synergistic treatment specifically comprises: first, low-temperature extrusion at a temperature of 60-70°C and a pressure of 15-20MPa; and then ultrasonic treatment at 200-300W for 30-40min.

[0021] In a preferred embodiment, in step (1), in the soybean meal pretreatment liquor, the crude fiber dissociation rate is above 40% and the phytic acid destruction rate is above 80%; the composition of the soybean meal pretreatment liquor is: soluble sugar >7.0g / L and free amino acid >1.0g / L.

[0022] In the present application, the low-temperature extrusion treatment can effectively destroy the fiber structure of soybean meal and the binding site of anti-nutritional factors, assist the ultrasonic treatment, and further promote the dissolution of protein and soluble sugar in soybean meal, thereby effectively improving the dissolution rate of crude fiber, the destruction rate of phytic acid, and the dissolution rate of xylan while avoiding traditional enzymatic treatment.

[0023] In a preferred embodiment, in step (1), the screw configuration of the low-temperature extrusion equipment adopts a three-section design of conveying section-compression section-shearing section; preferably, three groups of reverse threads are arranged in the shearing section.

[0024] In a preferred embodiment, in step (1), during the low-temperature extrusion treatment, the screw rotation speed is 100-120 r / min.

[0025] In a preferred embodiment, in step (1), during the ultrasonic treatment, the ultrasonic frequency is 25-30 kHz.

[0026] In a preferred embodiment, in step (2), the sterilization conditions are 115°C, 0.1 MPa, and sterilization for 20-25 min.

[0027] In a preferred embodiment, in step (3), the complex bacterial population comprises Lactobacillus plantarum (viable bacterial count ≥1.0×10 0 CFU / mL), Saccharomyces cerevisiae (viable bacterial count ≥5.0×10 9 CFU / mL), and Bacillus coagulans (viable bacterial count ≥2.0×10 9 CFU / mL) at a volume ratio of 2:(0.5-1.5):(0.5-1.5).

[0028] In a preferred embodiment, in step (3), after the nitrogen gas is introduced in the first stage, the oxygen concentration is ≤0.5%; after the pH of the system in the first stage is reduced to 4.0-4.5, the growth of miscellaneous bacteria can be effectively inhibited.

[0029] In a preferred embodiment, in step (3), the enzyme activity enhancer is composed of L-cysteine hydrochloride and vitamin B6 at a mass ratio of (1-3):1.

[0030] In the enzyme activity enhancer of the present application, L-cysteine hydrochloride can protect the sulfhydryl active site of protease, vitamin B6 can promote the metabolic pathway of protease synthesis by bacteria as a coenzyme, and the two can synergistically effectively improve the protease activity.

[0031] In a preferred embodiment, in step (3), the protease activity in the fermentation broth is ≥320 U / mL, and the total number of viable bacteria is ≥1.2×10 0 CFU / mL.

[0032] In a preferred embodiment, in step (4), the centrifugation conditions include centrifugation at 4℃, 7000-9000r / min for 15-20min.

[0033] Another object of the present application is to provide a proteinase beverage prepared by any of the above methods.

[0034] Compared with the prior art, the technical scheme of the present application has the following advantages:

[0035] 1. In the prior art, single physical crushing or enzymatic hydrolysis process is usually used for pretreatment of plant meal (such as soybean meal, corn meal); single physical crushing cannot effectively break the high fiber structure and anti-nutritional factors of the cyperus esculeus meal, and the enzymatic hydrolysis process is easy to introduce exogenous enzyme residues, which not only increases the cost but also may affect the safety of the product.

[0036] Therefore, the present application innovatively proposes a low-temperature extrusion and ultrasonic synergistic pretreatment scheme: first, low-temperature extrusion is carried out at a temperature of 60-70℃ and a pressure of 15-20MPa to break the fiber bundle structure of the cyperus esculeus meal and dissociate the binding sites of phytic acid and protein; then, 200-300W ultrasonic treatment is used for 30-40min to further break the fiber fragments after extrusion through cavitation effect and mechanical vibration, promote the dissolution of protein and soluble sugar, and destroy the molecular structure of phytic acid, thereby effectively improving the crude fiber dissociation rate, phytic acid destruction rate and xylan dissolution rate.

[0037] 2. In the present application, by using food-grade specific complex flora, corresponding two-stage temperature control fermentation process and enzyme activity enhancer, the protease activity and total number of viable bacteria at the end of fermentation are effectively improved.

[0038] Specifically: Lactobacillus plantarum can quickly produce acid in the anaerobic stage to reduce the pH of the system to 4.0-4.5 and inhibit the growth of miscellaneous bacteria; Saccharomyces cerevisiae can secrete amylase and saccharifying enzyme to convert soluble sugar in the soybean meal pretreatment liquid and arabinose dissolved from xylan into small molecule sugars, providing carbon source for Bacillus coagulans to produce enzymes; Bacillus coagulans dominates the synthesis of protease in the micro-aerobic stage, and its high temperature resistance and gastric acid resistance characteristics can improve the adaptability of the product in the human intestinal tract. In addition, the addition of 0.1-0.2g enzyme activity enhancer per 100mL of fermentation liquid can not only protect the sulfhydryl active site of protease, but also serve as a coenzyme to promote the metabolic pathway of the flora to synthesize protease, thereby improving the protease activity.

[0039] 3. The fermentation process provided by the present application is simple and convenient to handle, the equipment used is conventional food industry equipment, and is suitable for large-scale industrial production. At the same time, the protease activity in the prepared fermentation liquid is ≥320U / mL, and the total number of viable bacteria is ≥1.2×10 0CFU / mL, the optimum pH of protease (3.8-4.8), the optimum temperature (37-39 DEG C), the activity retention rate in stomach is 65%, the activity retention rate in small intestine is 88%, and the human gastrointestinal environment is highly adapted, and has broad industrialization prospects. DETAILED DESCRIPTION

[0040] In order to make the person skilled in the art better understand the present application, the present application will be further described in detail below in combination with specific embodiments, but it should be understood that the protection scope of the present application is not limited by the specific embodiments, and all other embodiments obtained by the person skilled in the art without creative labor based on the examples in the present application belong to the protection scope of the present application.

[0041] If not specifically indicated, the technical means used in the present application is the conventional means familiar to those skilled in the art, and various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by the existing method. The reagents used in the present application are analytical pure unless otherwise specified.

[0042] The test method used in the embodiments of the present application includes:

[0043] 1. Crude fiber dissociation rate: take 1.0g of residue after pretreatment (centrifugal separation, 45 DEG C drying to constant weight), add 200mL of boiling 0.5mol / L H2SO4, reflux for 30min, filter while hot; the residue is washed to neutral with hot water, 200mL of boiling 0.5mol / L NaOH is added, refluxed for 30min, and then filtered and washed to neutral with hot water; the residue is dried to constant weight at 105 DEG C (recorded as m1), and then burned to constant weight at 550 DEG C (recorded as m2);

[0044] Calculation formula: crude fiber dissociation rate (%) = (crude fiber content before treatment - (m1-m2) / 1.0) / crude fiber content before treatment x 100%; the crude fiber content of corylus avellana meal before treatment is 8.5%.

[0045] 2. Phytic acid destruction rate:

[0046] (1) Pretreatment liquid sample preparation: take 10mL of pretreatment liquid, add 5mL of 0.5mol / L HCl, oscillate for 30min, centrifuge at 10000r / min for 15min, and filter the supernatant through a 0.22μm filter membrane;

[0047] (2) Chromatographic conditions: C18 column (250mm x 4.6mm), mobile phase is 0.01mol / L KH2PO4 (pH3.0), flow rate is 1.0mL / min, column temperature is 30 DEG C, and detection wavelength is 210nm;

[0048] (3) Standard curve drawing: prepare 0.1-10 μg / mL phytic acid standard solution, determine the peak area and draw the standard curve;

[0049] Calculation formula: phytic acid destruction rate (%) = (phytic acid content before treatment - sample phytic acid content) / phytic acid content before treatment x 100%; The phytic acid content of cyperus esculentus meal before treatment is taken as 1.2%.

[0050] 3. Pretreatment liquid soluble sugar determination:

[0051] (1) Sample preparation: take 5 mL of pretreatment liquid, add 10 mL of anhydrous ethanol, shake for 20 min, centrifuge at 8000 r / min for 10 min, and then dilute the supernatant to 25 mL with ultrapure water, and filter through a 0.22 μm filter membrane;

[0052] (2) Chromatographic conditions: amino column (250 mm x 4.6 mm), mobile phase is acetonitrile: water = 75:25 (v / v), flow rate is 1.0 mL / min, column temperature is 35°C, and differential refractive index detector is used;

[0053] (3) Standard curve drawing: prepare glucose standard solution (1-10 g / L), determine the peak area and calculate the total amount of soluble sugar.

[0054] 4. Pretreatment liquid free amino acid determination:

[0055] (1) Sample preparation: take 10 mL of pretreatment liquid, add 5 mL of sulfosalicylic acid (5%) to precipitate protein, centrifuge at 12000 r / min for 20 min, filter the supernatant with a 0.22 μm filter membrane, and adjust the pH to 2.2;

[0056] (2) Instrument conditions: cation exchange column, column temperature 50°C, ninhydrin coloration, detection wavelength 570 nm (proline 440 nm);

[0057] (3) Calculation: directly determine the total amount of free amino acids (calibrated with glutamic acid as standard) by amino acid automatic analyzer.

[0058] Example 1

[0059] A method for preparing a protease beverage from cyperus esculentus powder fermentation, specifically comprising the following steps:

[0060] (1) Directional pretreatment:

[0061] The cold-pressed oil of Cyperus esculentus L. was collected within 24 hours, and the Cyperus esculentus L. meal (moisture 10.5%, protein 8.3%) was cleaned by a 1.0 mm vibrating screen and magnetic separation, and the purity was 99.2%. The Cyperus esculentus L. meal was ground to 100 mesh, and mixed with deionized water at a solid-liquid ratio of 1 g:9 mL. The mixture was first extruded at 65 DEG C and 18 MPa (screw speed 110 r / min), and then ultrasonically treated at 250 W and 28 kHz for 35 min to obtain a Cyperus esculentus L. meal pretreatment liquid.

[0062] The obtained pretreatment liquid was detected, and the results were as follows: the crude fiber dissociation rate was 45%, and the phytic acid destruction rate was 83%; the composition of the pretreatment liquid was as follows: soluble sugar 8.2 g / L, free amino acid 1.3 g / L.

[0063] (2) Preparation of fermentation medium:

[0064] 2.5 g of skimmed milk powder, 0.7 g of glucose, and 0.15 g of KH2PO4 were added to 100 mL of the pretreatment liquid, the pH was adjusted to 6.2, and the mixture was sterilized at 115 DEG C and 0.1 MPa for 22 min, and then cooled to 36 DEG C.

[0065] (3) Subsection fermentation:

[0066] The compound bacterial community (Lactobacillus plantarum, 1.2 x 10 0 CFU / mL; Saccharomyces cerevisiae, 6.0 x 10 9 CFU / mL; Bacillus coagulans, 2.5 x 10 9 CFU / mL) was inoculated at 6% (v / v).

[0067] The first stage was anaerobic (nitrogen was introduced, and the oxygen concentration was 0.3%) fermentation at 36 DEG C for 20 h, and the pH was reduced to 4.2; the second stage was fermentation at 39 DEG C for 14 h, sterile air containing 6% oxygen was introduced, and enzyme activity enhancer (L-cysteine hydrochloride and vitamin B6 were mixed at a mass ratio of 2:1) was added at 0.15 g / 100 mL.

[0068] The obtained fermentation liquid was detected, and the results were as follows: the protease activity was 350 U / mL, and the total number of viable bacteria was 1.5 x 10 0 CFU / mL.

[0069] (4) Formulation:

[0070] The obtained fermentation liquid was centrifuged at 4 DEG C and 8000 r / min for 18 min, the supernatant was filtered through a 0.22 mu m filter membrane, 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol were added, and the mixture was homogenized at 22 MPa and 42 DEG C for 2 times. After instant sterilization at 135 DEG C for 4 s, the mixture was aseptically filled within 8 s, and stored at 2-8 DEG C.

[0071] Example 2

[0072] A method for preparing a protease beverage from Cyperus esculentus L. powder fermentation, specifically comprising the following steps:

[0073] (1) Directional pretreatment:

[0074] Select Cyperus esculentus L. meal within 48 hours after cold pressing (moisture 11.8%, protein 8.5%), purity 99.5% after cleaning. Ultrafine grinding to 80 mesh, mixing with deionized water according to solid-liquid ratio 1g:8mL, first low-temperature extrusion (screw speed 100r / min) at 60℃, 15MPa, then ultrasonic treatment at 200W, 25kHz for 30min.

[0075] Detect the obtained pretreatment, the results are: crude fiber dissociation rate 40%, phytic acid destruction rate 80%; pretreatment liquid composition: soluble sugar 7.1g / L, free amino acid 1.0g / L.

[0076] (2) Preparation of fermentation medium:

[0077] Add 2.0g skim milk powder, 1.0g glucose, 0.1g KH2PO4 per 100mL pretreatment liquid, adjust pH to 6.0, sterilize at 115℃ for 25min, cool to 35℃.

[0078] (3) Subsection fermentation:

[0079] Inoculate the complex flora (Lactobacillus plantarum, 1.2×10 0 CFU / mL; Saccharomyces cerevisiae, 6.0×10 9 CFU / mL; Bacillus coagulans, 2.5×10 9 CFU / mL, volume ratio 2:0.5:0.5) at 5% (v / v).

[0080] The first stage is anaerobic (nitrogen gas is introduced, oxygen concentration is 0.5%) fermentation at 35℃ for 24h, pH drops to 4.5; the second stage is to increase the temperature to 38℃, introduce sterile air containing 5% oxygen, add enzyme activity enhancer (L-cysteine hydrochloride and vitamin B6, mass ratio 1:1) at 0.1g / 100mL, continue to ferment for 16h.

[0081] Detect the obtained fermentation liquid, the results are: protease activity 320U / mL, total number of viable bacteria 1.2×10 0 CFU / mL

[0082] (4) Formulation:

[0083] The obtained fermentation liquid was centrifuged at 4°C and 7000r / min for 20min, 0.5g / 100mL fructooligosaccharide and 1.0g / 100mL erythritol were added, and the mixture was homogenized at 20MPa and 40°C for 3 times; then the mixture was instantaneously sterilized at 135°C for 4s, and then was aseptically filled within 8s, and was stored at 2-8°C.

[0084] Example 3

[0085] A method for preparing a protease beverage from Cyperus esculentus L. powder fermentation, specifically comprising the following steps:

[0086] (1) Directional pretreatment:

[0087] The Cyperus esculentus L. meal within 36h after cold pressing was selected (moisture 10.2%, protein 9.8%), and the purity was 99.1% after cleaning. The meal was ultra-micronized to 120 meshes, mixed with deionized water at a solid-liquid ratio of 1g:10mL, and then was low-temperature extruded at 70°C and 20MPa (screw rotation speed 120r / min), and then was ultrasonically treated at 300W and 30kHz for 40min.

[0088] The obtained pretreatment was detected, and the results were as follows: coarse fiber dissociation rate 48%, phytic acid destruction rate 85%; pretreatment liquid composition: soluble sugar 8.8g / L, free amino acid 1.5g / L.

[0089] (2) Preparation of fermentation medium:

[0090] 3.0g skim milk powder, 1.1g glucose and 0.2g KH2PO4 were added to 100mL of the pretreatment liquid, the pH was adjusted to 6.5, and the mixture was sterilized at 115°C for 20min, and then was cooled to 38°C.

[0091] (3) Subsection fermentation:

[0092] The compound bacterial flora (Lactobacillus plantarum, 1.2×10 0 CFU / mL; Saccharomyces cerevisiae, 6.0×10 9 CFU / mL; Bacillus coagulans, 2.5×10 9 CFU / mL, volume ratio 2:1.5:1.5) was inoculated at 8% (v / v).

[0093] The first stage was anaerobic (nitrogen was introduced, oxygen concentration 0.2%) fermentation at 37°C for 18h, and the pH was reduced to 4.0; the second stage was continued fermentation at 40°C by introducing sterile air containing 8% oxygen, and 0.2g / 100mL of enzyme activity enhancer (L-cysteine hydrochloride and vitamin B6 were composed at a mass ratio of 3:1) was added.

[0094] The obtained fermentation liquid was detected, and the results were as follows: protease activity 380U / mL, total number of living bacteria 1.8×10 0 CFU / mL

[0095] (4) Formulation:

[0096] The obtained fermentation liquid was centrifuged at 4°C and 9000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane; 1.0 g / 100 mL of fructooligosaccharide and 1.5 g / 100 mL of erythritol were added, and homogenized twice at 25 MPa and 45°C; then, after instant sterilization at 135°C for 4 s, sterile filling was performed within 8 s, and cold storage was performed at 2-8°C.

[0097] Comparative Example 1 (without low-temperature extrusion and ultrasonic synergistic treatment)

[0098] A method for preparing a protease beverage from Cyperus esculentus L. powder fermentation, specifically comprising the following steps:

[0099] (1) Directional pretreatment:

[0100] Select Cyperus esculentus meal within 24 h after cold pressing oil (moisture 10.5%, protein 8.3%), clean it through a 1.0 mm vibrating screen and magnetic separation, and the purity is 99.2%. Ultrafine grinding to 100 mesh, mix with deionized water at a solid-liquid ratio of 1 g:9 mL to obtain a Cyperus esculentus meal pretreatment liquid.

[0101] Detect the obtained pretreatment, and the results are: crude fiber dissociation rate 22%, phytic acid destruction rate 45%; soluble sugar in the pretreatment liquid 4.5 g / L, free amino acid 0.6 g / L.

[0102] (2) Preparation of fermentation medium:

[0103] Add 2.5 g of skim milk powder, 0.7 g of glucose, and 0.15 g of KH2PO4 to 100 mL of the pretreatment liquid, adjust the pH to 6.2, sterilize at 115°C and 0.1 MPa for 22 min, and cool to 36°C.

[0104] (3) Subsection fermentation:

[0105] Inoculate the complex flora (Lactobacillus plantarum, 1.2×10 0 CFU / mL; Saccharomyces cerevisiae, 6.0×10 9 CFU / mL; Bacillus coagulans, 2.5×10 9 CFU / mL, volume ratio 2:1:1) at 6% (v / v).

[0106] The first stage is anaerobic (nitrogen gas is introduced, oxygen concentration is 0.3%) fermentation at 36°C for 20 h, and the pH is reduced to 4.2; the second stage is to increase the temperature to 39°C, introduce sterile air containing 6% oxygen, and add enzyme activity enhancer (L-cysteine hydrochloride and vitamin B6 in a mass ratio of 2:1) at 0.15 g / 100 mL, and continue to ferment for 14 h.

[0107] The obtained fermentation liquor was detected, and the results were as follows: the protease activity of the fermentation liquor was 180 U / mL, the viable cell count was 8.0 x 10 9 CFU / mL.

[0108] (4) Formulation:

[0109] The obtained fermentation liquor was centrifuged at 4 DEG C. and 8000 r / min for 18 min, the supernatant was filtered through a 0.22 mu m filter membrane, 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol were added, and homogenization was performed twice at 22 MPa and 42 DEG C. After instant sterilization at 135 DEG C. for 4 s, sterile filling was performed within 8 s, and cold storage was performed at 2-8 DEG C.

[0110] Comparative Example 2 (fermentation of a single Lactobacillus plantarum)

[0111] A method for preparing a protease beverage by fermentation of Cyperus esculentus powder, specifically comprising the following steps:

[0112] (1) Directional pretreatment:

[0113] Cyperus esculentus meal (moisture 10.5%, protein 8.3%) within 24 h after cold pressing was selected, cleaned through a 1.0 mm vibrating screen and magnetic separation, and the purity was 99.2%. The meal was ultra-finely ground to 100 meshes, mixed with deionized water at a solid-liquid ratio of 1 g:9 mL, first subjected to low-temperature extrusion at 65 DEG C. and 18 MPa (screw rotation speed 110 r / min), and then subjected to ultrasonic treatment at 250 W and 28 kHz for 35 min to obtain a meal pretreatment liquor.

[0114] The obtained pretreatment was detected, and the results were as follows: the crude fiber dissociation rate was 45%, and the phytic acid destruction rate was 83%; the composition of the pretreatment liquor was as follows: soluble sugar 8.2 g / L, free amino acid 1.3 g / L.

[0115] (2) Preparation of fermentation medium:

[0116] 2.5 g of skim milk powder, 0.7 g of glucose, and 0.15 g of KH2PO4 were added to 100 mL of the pretreatment liquor, the pH was adjusted to 6.2, sterilization was performed at 115 DEG C. and 0.1 MPa for 22 min, and the temperature was cooled to 36 DEG C.

[0117] (3) Subsection fermentation:

[0118] Lactobacillus plantarum (1.2 x 10 0 CFU / mL) was inoculated at 6% (v / v).

[0119] The first stage is anaerobic fermentation (nitrogen is introduced, and the oxygen concentration is 0.3%) at 36°C for 20 hours, and the pH is reduced to 3.8; the second stage is to increase the temperature to 39°C, introduce sterile air containing 6% oxygen, and add enzyme activity enhancer (L-cysteine hydrochloride and vitamin B6, which are composed of 2:1 by mass) at 0.15 g / 100 mL, and continue to ferment for 14 hours.

[0120] The obtained fermentation liquor is detected, and the results are as follows: the protease activity is 140 U / mL, the total number of viable bacteria is 9.0 x 10 9 CFU / mL. In addition, the obtained fermentation liquor is too sour and has poor palatability.

[0121] (4) Formulation:

[0122] The obtained fermentation liquor is centrifuged at 4°C and 8000 r / min for 18 min, the supernatant is filtered through a 0.22 μm filter membrane, 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol are added, and homogenization is performed twice at 22 MPa and 42°C. After instant sterilization at 135°C for 4 s, sterile filling is performed within 8 s, and cold storage is performed at 2-8°C.

[0123] Comparative Example 3 (single fermentation of Saccharomyces cerevisiae)

[0124] A method for preparing a protease beverage from Cyperus esculentus powder fermentation, specifically comprising the following steps:

[0125] (1) Directional pretreatment:

[0126] The Cyperus esculentus meal within 24 hours after cold pressing oil (moisture 10.5%, protein 8.3%) is cleaned by a 1.0 mm vibrating screen and magnetic separation, and the purity is 99.2%. The meal is ultra-finely ground to 100 mesh, mixed with deionized water at a solid-liquid ratio of 1 g:9 mL, first low-temperature extruded at 65°C and 18 MPa (screw rotation speed 110 r / min), and then ultrasonically treated at 250 W and 28 kHz for 35 min to obtain a meal pretreatment liquid.

[0127] The obtained pretreatment is detected, and the results are as follows: the crude fiber dissociation rate is 45%, and the phytic acid destruction rate is 83%; the composition of the pretreatment liquid is: soluble sugar 8.2 g / L, free amino acid 1.3 g / L.

[0128] (2) Preparation of fermentation medium:

[0129] 2.5 g of skim milk powder, 0.7 g of glucose, and 0.15 g of KH2PO4 are added to 100 mL of the pretreatment liquid, the pH is adjusted to 6.2, sterilized at 115°C and 0.1 MPa for 22 min, and cooled to 36°C.

[0130] (3) Subsection fermentation:

[0131] Inoculate with 6% (v / v) of Saccharomyces cerevisiae (6.0 × 10⁻⁶). 9 (CFU / mL).

[0132] In the first stage, anaerobic fermentation was carried out at 36℃ (nitrogen was introduced, oxygen concentration was 0.3%) for 20 hours, and the pH dropped to 5.8. In the second stage, the temperature was raised to 39℃, sterile air containing 6% oxygen was introduced, and an enzyme activity enhancer (composed of L-cysteine ​​hydrochloride and vitamin B6 in a mass ratio of 2:1) was added at 0.15g / 100mL, and fermentation continued for 14 hours.

[0133] The fermentation broth was tested, and the results were as follows: protease activity 80 U / mL, total viable cell count 6.5 × 10⁻⁶. 9 CFU / mL. Additionally, the resulting fermentation broth had a distinct yeasty smell and a slightly astringent taste.

[0134] (4) Allocation:

[0135] The obtained fermentation broth was centrifuged at 8000 r / min for 18 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane. 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol were added, and the mixture was homogenized twice at 22 MPa and 42℃. After being sterilized at 135℃ for 4 seconds, the mixture was aseptically filled within 8 seconds and refrigerated at 2-8℃.

[0136] Comparative Example 4 (fermentation by a single Bacillus coagulans)

[0137] A method for preparing a protease beverage by fermenting tiger nut powder specifically includes the following steps:

[0138] (1) Targeted pretreatment:

[0139] Oilseed meal (10.5% moisture, 8.3% protein) within 24 hours after cold pressing was selected, cleaned by a 1.0mm vibrating sieve and magnetic separation, achieving a purity of 99.2%. It was then ultra-finely pulverized to 100 mesh, mixed with deionized water at a solid-liquid ratio of 1g:9mL, and first subjected to low-temperature extrusion at 65℃ and 18MPa (screw speed 110r / min). Following this, it was ultrasonically treated at 250W and 28kHz for 35min to obtain a pretreated soybean meal solution.

[0140] The results of the pretreatment were as follows: crude fiber dissociation rate 45%, phytic acid destruction rate 83%; pretreatment solution composition: soluble sugar 8.2 g / L, free amino acids 1.3 g / L.

[0141] (2) Preparation of fermentation culture medium:

[0142] Add 2.5g skim milk powder, 0.7g glucose, and 0.15g KH2PO4 to every 100mL of pretreatment solution, adjust the pH to 6.2, sterilize at 115℃ and 0.1MPa for 22min, and cool to 36℃.

[0143] (3) Segmented fermentation:

[0144] Inoculate with 6% (v / v) Bacillus coagulans (2.5 × 10⁻⁶). 9 (CFU / mL).

[0145] In the first stage, anaerobic fermentation was carried out at 36℃ (nitrogen was introduced, oxygen concentration was 0.3%) for 20 hours, and the pH dropped to 5.5. In the second stage, the temperature was raised to 39℃, sterile air containing 6% oxygen was introduced, and an enzyme activity enhancer (composed of L-cysteine ​​hydrochloride and vitamin B6 in a mass ratio of 2:1) was added at 0.15g / 100mL, and fermentation continued for 14 hours.

[0146] The fermentation broth was tested, and the results were as follows: protease activity 160 U / mL, total viable count 8.0 × 10⁻⁶. 9 CFU / mL. Additionally, the resulting fermentation broth had a bland flavor and lacked any fermented aroma.

[0147] (4) Allocation:

[0148] The obtained fermentation broth was centrifuged at 8000 r / min for 18 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane. 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol were added, and the mixture was homogenized twice at 22 MPa and 42℃. After being sterilized at 135℃ for 4 seconds, the mixture was aseptically filled within 8 seconds and refrigerated at 2-8℃.

[0149] Comparative Example 5 (without enzyme synergist)

[0150] A method for preparing a protease beverage by fermenting tiger nut powder specifically includes the following steps:

[0151] (1) Targeted pretreatment:

[0152] Oilseed meal (10.5% moisture, 8.3% protein) within 24 hours after cold pressing was selected, cleaned by a 1.0mm vibrating sieve and magnetic separation, achieving a purity of 99.2%. It was then ultra-finely pulverized to 100 mesh, mixed with deionized water at a solid-liquid ratio of 1g:9mL, and first subjected to low-temperature extrusion at 65℃ and 18MPa (screw speed 110r / min). Following this, it was ultrasonically treated at 250W and 28kHz for 35min to obtain a pretreated soybean meal solution.

[0153] The results of the pretreatment were as follows: crude fiber dissociation rate 45%, phytic acid destruction rate 83%; pretreatment solution composition: soluble sugar 8.2 g / L, free amino acids 1.3 g / L.

[0154] (2) Preparation of fermentation culture medium:

[0155] Add 2.5g skim milk powder, 0.7g glucose, and 0.15g KH2PO4 to every 100mL of pretreatment solution, adjust the pH to 6.2, sterilize at 115℃ and 0.1MPa for 22min, and cool to 36℃.

[0156] (3) Segmented fermentation:

[0157] Inoculate with a 6% (v / v) co-culture of Lactobacillus plantarum (1.2 × 10¹) 0 CFU / mL; Saccharomyces cerevisiae, 6.0 × 10⁻⁶ 9 CFU / mL; Bacillus coagulans, 2.5 × 10⁻⁶ 9 CFU / mL, volume ratio 2:1:1).

[0158] The first stage involves anaerobic fermentation at 36℃ (nitrogen gas introduced, oxygen concentration 0.3%) for 20 hours, during which the pH drops to 4.2. The second stage involves raising the temperature to 39℃, introducing sterile air containing 6% oxygen, and continuing fermentation for another 14 hours.

[0159] The fermentation broth was tested, and the results were as follows: protease activity was 290 U / mL, a decrease of 17% compared to Example 1; viable cell count was 1.4 × 10¹. 0 CFU / mL.

[0160] (4) Allocation:

[0161] The obtained fermentation broth was centrifuged at 8000 r / min for 18 min at 4℃, and the supernatant was filtered through a 0.22 μm filter membrane. 0.8 g / 100 mL of fructooligosaccharide and 1.2 g / 100 mL of erythritol were added, and the mixture was homogenized twice at 22 MPa and 42℃. After being sterilized at 135℃ for 4 seconds, the mixture was aseptically filled within 8 seconds and refrigerated at 2-8℃.

[0162] Application examples

[0163] The optimal pH and optimal temperature for the fermentation enzyme prepared in Example 1 were verified, including the following steps:

[0164] (a) Determination of the optimal pH for protease

[0165] (1) Reagents used

[0166] Substrate: 1% (w / v) casein solution (prepared with different pH buffers);

[0167] Buffer system: pH 2.0 (0.1 mol / L citrate-sodium citrate), pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0 (all 0.1 mol / L citrate-sodium citrate buffer).

[0168] Enzyme solution: Take the supernatant of the fermentation broth from Example 1 (filtered through a 0.22 μm filter membrane) and dilute it 10 times with the corresponding pH buffer (to ensure that the enzyme activity is within the detection linear range).

[0169] Folin-Ciocalteu reagent (1:5 dilution), 0.4 mol / L Na₂CO₃ solution.

[0170] (2) Experimental steps

[0171] 1. Take 10 test tubes and add 1 mL of 1% casein solution with different pH values ​​to each tube. Incubate in a 37°C water bath for 10 min (simulating gastrointestinal temperature).

[0172] 2. Add 1 mL of diluted enzyme solution to each test tube, shake well, and react in a water bath at 37°C for 10 min;

[0173] 3. Immediately add 2 mL of 0.4 mol / L trichloroacetic acid (to terminate the reaction), shake well, let stand for 10 min, and centrifuge at 4000 r / min for 10 min;

[0174] 4. Take 1 mL of supernatant, add 5 mL of 0.4 mol / L Na2CO3 solution, then add 1 mL of Folin reagent, and develop the color in a 40℃ water bath for 20 min;

[0175] 5. Using "buffer solution + casein + trichloroacetic acid + enzyme solution" as a blank control, the absorbance value was measured at 680 nm;

[0176] 6. Calculate the protease activity (unit: U / mL) based on the absorbance value. The activity calculation formula is:

[0177] Protease activity (U / mL) = (A-A0)×K×N (A: sample absorbance, A0: blank absorbance, K: activity coefficient corresponding to the slope of the standard curve, N: dilution factor).

[0178] (3) Result determination

[0179] A curve was plotted with pH on the x-axis and protease activity on the y-axis. The pH range corresponding to the peak of the curve is the optimal pH. The optimal pH range of 3.8-4.8 was found to cover the pH after gastric dilution (3.0-4.5) and the pH of the upper small intestine (5.0-6.0), showing good gastrointestinal pH adaptability.

[0180] (II) Determination of the optimal temperature for protease

[0181] (1) Reagents used:

[0182] 1% (w / v) casein solution (prepared with pH 4.0 buffer, using the pH value with the highest activity in the optimal pH preliminary experiment); buffer: 0.1 mol / L citrate-sodium citrate buffer (pH 4.0); enzyme solution: the same diluted enzyme solution used in the optimal pH determination.

[0183] (2) Experimental steps

[0184] 1. Take 8 test tubes, add 1 mL of 1% casein solution with pH 4.0 to each tube, and incubate them in water baths at 25℃, 30℃, 35℃, 37℃, 38℃, 40℃, 45℃, and 50℃ for 10 min respectively;

[0185] 2. Add 1 mL of diluted enzyme solution to each test tube, shake well, and react at the corresponding temperature for 10 min;

[0186] 3. Subsequent steps (termination of reaction, color development, absorbance measurement) are the same as for the optimal pH determination;

[0187] 4. Calculate the protease activity at different temperatures and plot a curve with temperature on the x-axis and activity on the y-axis.

[0188] (3) Result determination

[0189] The temperature range corresponding to the peak of the curve is 37-39℃, which perfectly matches the temperature of the human gastrointestinal tract (37-38℃).

[0190] (III) Verification of gastrointestinal environment adaptability (in vitro simulated digestion experiment)

[0191] (1) Experimental design

[0192] The digestion process of the beverage after it enters the human body was simulated: it was first diluted in the stomach (pH 3.5) and then entered the small intestine (pH 4.5-5.0). The temperature was maintained at 37°C, and the activity retention rate of the protease was measured during this process.

[0193] (2) Steps

[0194] 1. Gastric simulation: Take 1 mL of enzyme solution from Example 1, add 4 mL of pH 3.5 buffer (simulating gastric juice dilution, final pH 3.5), incubate at 37°C for 30 min, and measure the enzyme activity at this time (denoted as A1).

[0195] 2. Small intestinal simulation: Take 1 mL of the above gastric simulation solution, add 4 mL of pH 4.8 buffer (simulating the small intestinal environment), incubate at 37℃ for 60 min, and measure enzyme activity (denoted as A2).

[0196] 3. Blank control: The enzyme activity was measured directly at pH 7.0 and 37℃ (denoted as A0).

[0197] 4. Calculation formula: Gastric viable retention rate = (A1 / A0) × 100%;

[0198] Small intestinal viability retention rate = (A2 / A0) × 100%.

[0199] (3) Result determination

[0200] Tests showed that the activity retention rate in the stomach was 65% and in the small intestine was 88%, indicating that the prepared protease is highly compatible with the gastrointestinal environment and is more suitable for drinking.

[0201] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing a protease beverage by fermenting tiger nut powder, characterized in that, Includes the following steps: (1) Targeted pretreatment: Soybean meal within 48 hours after cold pressing of tiger nuts for oil extraction was selected, ultra-finely pulverized, and then added to deionized water. The mixture was then subjected to low-temperature extrusion and ultrasonic treatment to obtain a soybean meal pretreatment solution. (2) Preparation of fermentation culture medium: Add 2-3g of skim milk powder, 1-1.2g of glucose, and 0.1-0.2g of KH2PO4 to every 100mL of soybean meal pretreatment solution obtained in step (1), adjust the pH to 6.0-6.5, sterilize, and cool to 35-38℃; (3) Segmented fermentation: In the fermentation medium prepared in step (2), inoculate the complex microbial community at 5-8%. In the first stage, introduce nitrogen gas and anaerobic ferment at 35-37℃ for 18-24 hours to reduce the pH of the system to 4.0-4.

5. In the second stage, raise the temperature to 38-40℃, introduce sterile air containing 5-8% oxygen, add enzyme activity enhancer at 0.1-0.2 g / 100 mL, and continue fermentation for 12-16 hours. After fermentation is completed, collect the fermentation broth.

2. The method for preparing a protease beverage by fermenting tiger nut powder as described in claim 1, characterized in that, Step (3) is followed by (4) preparation: Centrifuge the fermentation broth obtained in step (3), and filter the supernatant through a 0.22 μm organic filter membrane; add 0.5-1.0 g / 100 mL of fructooligosaccharides and 1-1.5 g / 100 mL of erythritol, homogenize 2-3 times at 20-25 MPa and 40-45℃, sterilize at 135℃ for 3-5 seconds, and complete aseptic filling within 10 seconds. Refrigerate at 2-8℃ to obtain the protease beverage.

3. The method for preparing a protease beverage from tiger nut powder by fermentation as described in claim 1, characterized in that, In step (1), the process parameters for cold pressing oil extraction from tiger nuts are: pressing temperature ≤60℃, pressing pressure 30-40Mpa; the resulting soybean meal has a moisture content ≤12%, protein content 5-15%, and purity ≥99.0%.

4. The method for preparing a protease beverage by fermenting tiger nut powder as described in claim 1, characterized in that, In step (1), the particle size after ultrafine grinding is 80-120 mesh; The solid-liquid ratio of soybean meal to deionized water is 1g:(1-12)mL.

5. The method for preparing a protease beverage from tiger nut powder by fermentation as described in claim 1, characterized in that, In step (1), the low-temperature extrusion and ultrasonic synergistic treatment specifically includes: firstly, low-temperature extrusion is carried out at a temperature of 60-70℃ and a pressure of 15-20MPa; then, ultrasonic treatment at 200-300W is performed for 30-40 minutes.

6. The method for preparing a protease beverage from tiger nut powder by fermentation as described in claim 5, characterized in that, In step (1), during the low-temperature extrusion process, the screw speed is 100-120 r / min; During the ultrasonic treatment, the ultrasonic frequency is 25-30kHz.

7. The method for preparing a protease beverage from tiger nut powder by fermentation as described in claim 1, characterized in that, In step (3), the complex microbial community consists of Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus coagulans in a volume ratio of 2:(0.5-1.5):(0.5-1.5).

8. The method for preparing a protease beverage by fermenting tiger nut powder as described in claim 1, characterized in that, In step (3), the enzyme activator is composed of L-cysteine ​​hydrochloride and vitamin B6 in a mass ratio of (1-3):

1.

9. The method for preparing a protease beverage by fermenting tiger nut powder as described in claim 1, characterized in that, In step (3), the fermentation broth contains a protease activity ≥320 U / mL and a total viable count ≥1.2 × 10¹. 0 CFU / mL.

10. The protease beverage prepared by the method according to any one of claims 1-9.